PROJECT SUMMARY The discovery that regulatory RNAs control almost every biological pathway has revolutionized our understanding of gene expression over the past decade. At the forefront, microRNAs (miRNAs) have proven to be an abundant and essential class of RNA molecules in plants and animals. The importance of miRNAs in human biology is highlighted by the increasing recognition that misregulation of specific miRNA pathways contributes to complex diseases, including cancer, heart ailments and neuronal pathologies. MiRNAs depend on Argonaute (AGO) proteins to bind and regulate the expression of target genes at the post-transcriptional level. In C. elegans, the AGO Like Genes 1 and 2 (ALG-1, ALG-2) proteins are over 85% identical at the amino acid level and function redundantly during development. However, at adulthood these AGOs take on opposing roles, as alg- 1 or alg-2 mutants exhibit shortened or extended lifespans, respectively. Additionally, healthspan, as measured by maximum velocity, is reduced in alg-1 and increased in alg-2 mutants compared to wildtype adults. One longevity pathway that is differentially impacted by the loss of either AGO is Insulin/ IGF-1 Signaling (IIS). These preliminary studies lead to the hypothesis that differences in the expression or activity of alg-1 and alg-2 allow them to regulate distinct sets of genes in the IIS pathway to control lifespan in adult animals. In Aims 1 and 2, the genetic basis and potential tissue specific activities responsible for the opposing longevity functions of alg-1 and alg-2 will be determined. Cutting edge methods for detecting the specific miRNAs and targets bound by ALG-1 and ALG-2 will be utilized in Aim 3. This will result in the first map of miRNA specific target sites in an adult animal and provide unprecedented insights into the direct roles of alg-1 and alg-2 in the IIS, and perhaps other, longevity pathways. Furthermore, by revealing the miRNA targeting landscape in adult C. elegans a new foundation will be set for investigating longevity roles for conserved miRNAs and targets in other species. Another broad impact of this study is that it will reveal how two proteins considered redundant take on opposing roles in aging, which will provide a paradigm for considering the importance of context for the function of related genes in any pathway. Additionally, the methods used here to identify miRNA specific target sites are generally applicable across systems and, thus, will help eliminate a longstanding barrier to matching miRNAs with their endogenous target sites. Finally, a long-term goal of this work is to contribute to the rational design of strategies to modify gene expression with the intention of preventing or counteracting age-related maladies.